Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method

Justin A. Bishop, Xiao Jun Ma, Hongwei Wang, Yuling Luo, Peter B. Illei, Shanaz Begum, Janis M. Taube, Wayne M. Koch, William H. Westra

Research output: Contribution to journalArticle

171 Citations (Scopus)

Abstract

Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and polymerase chain reaction amplification-a challenging technique that is restricted to the research laboratory. The development of RNA in situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays containing 282 head and neck squamous cell carcinomas from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and an RNA ISH assay (RNAscope) targeting high-risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas in which the HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (ie, p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of an unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. p16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV-positive tumors exhibited p16 expression, whereas only 22 of 233 HPV-negative tumors were p16 positive (94% vs. 9%, P<0.0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16/HPV DNA), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.

Original languageEnglish (US)
Pages (from-to)1874-1882
Number of pages9
JournalAmerican Journal of Surgical Pathology
Volume36
Issue number12
DOIs
StatePublished - Dec 1 2012

Fingerprint

In Situ Hybridization
Messenger RNA
Carcinoma
Oropharynx
DNA
RNA
Carcinoma, squamous cell of head and neck
Surgical Pathology
Oncogene Proteins
DNA-Directed RNA Polymerases
Transcriptional Activation
Neoplasms
Neck
Immunohistochemistry
Head
Viruses
Polymerase Chain Reaction

Keywords

  • head and neck cancer
  • human papillomavirus
  • in situ hybridization
  • oropharyngeal cancer
  • p16
  • squamous cell carcinoma

ASJC Scopus subject areas

  • Anatomy
  • Surgery
  • Pathology and Forensic Medicine

Cite this

Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. / Bishop, Justin A.; Ma, Xiao Jun; Wang, Hongwei; Luo, Yuling; Illei, Peter B.; Begum, Shanaz; Taube, Janis M.; Koch, Wayne M.; Westra, William H.

In: American Journal of Surgical Pathology, Vol. 36, No. 12, 01.12.2012, p. 1874-1882.

Research output: Contribution to journalArticle

Bishop, Justin A. ; Ma, Xiao Jun ; Wang, Hongwei ; Luo, Yuling ; Illei, Peter B. ; Begum, Shanaz ; Taube, Janis M. ; Koch, Wayne M. ; Westra, William H. / Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. In: American Journal of Surgical Pathology. 2012 ; Vol. 36, No. 12. pp. 1874-1882.
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abstract = "Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and polymerase chain reaction amplification-a challenging technique that is restricted to the research laboratory. The development of RNA in situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays containing 282 head and neck squamous cell carcinomas from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and an RNA ISH assay (RNAscope) targeting high-risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas in which the HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (ie, p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17{\%}) HNSCCs including 43 of 77 (56{\%}) carcinomas from the oropharynx, 2 of 3 (67{\%}) metastatic HNSCCs of an unknown primary site, 2 of 7 (29{\%}) carcinomas from the sinonasal tract, and 2 of 195 (1{\%}) carcinomas from other head and neck sites. p16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV-positive tumors exhibited p16 expression, whereas only 22 of 233 HPV-negative tumors were p16 positive (94{\%} vs. 9{\%}, P<0.0001). There was also a high rate of concordance (99{\%}) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16/HPV DNA), the presence of E6/E7 transcripts was detected in 21 of 25 (84{\%}) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.",
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